Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications. We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney. Results in mice showed >300 days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400 days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models. We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.